Proposal Summary The goal of this project is to investigate the mechanism of incomplete cytokinesis that results in the formation of intercellular bridges in animal germline cells. These bridges, called ring canals in Drosophila, are integral to the formation of gametes as disruption of these bridges results in sterility. Incomplete cytokinesis events are widely observed in not only the germline but in some somatic tissues and are a hallmark of classical Hodgkin lymphoma providing evidence that incomplete cytokinesis events are relevant to non-germline biology. Using the Drosophila germline as a model for incomplete cytokinesis, the experiments proposed are designed to characterize the formation of ring canals in male and female germlines, elucidate the composition of proteins at the ring canal membrane via biochemical screens, and characterize novel ring canal components identified from these screens. The three integrated aims of this proposal will provide a comprehensive understanding of the cytoskeletal and cellular events that mediate ring canal formation, identify proteins that contribute to ring canal formation, and assess the function of novel proteins required for ring canal formation in the male and female germlines. Experiments proposed in Aim 1 will define the cellular events that mediate ring canal formation in the Drosophila germline. The mechanism of ring canal formation is unknown as there are no published studies examining ring canal formation with high-temporal resolution in the developing ovary or testis. Furthermore, the composition of ring canals differs between males and females suggesting there are varying strategies in the formation of ring canals between the sexes. Ultrastructural studies of ring canals reveal they are marked by an electron density at the membrane, however the components of this density are unknown. Using a combination of live cell imaging and electron tomography, ring canal formation and the accompanying changes in membrane electron density will be characterized. These experiments will be of great interest to researchers studying germline biology. Experiments proposed in Aim 2 will identify ring canal proteins that may facilitate ring canal formation. A proximity-dependent biotinylation proteomic approach coupled with isolation of intact ring canal complexes will reveal the substructure of ring canal complexes and identify proteins for further validation and characterization. The proteomes of males and females will be compared, allowing further insight into the shared features of ring canal formation between the germlines. In Aim 3, candidate proteins identified in either screen will be validated and characterized to determine whether they contribute to the mechanism of ring canal formation. Protein localization will first be assessed using antibodies or fluorescent tags, followed by genetic analyses to test for function. Identification of proteins that contribute to the inhibition of membrane abscission will be of interest to researchers studying incomplete cytokinesis in both germline and non-germline contexts.